Changes in intracellular Ca2+ concentration control a myriad of physiological processes including muscle contraction, secretion, mitosis, channel gating, chemotaxis and stomatal pore closure. During the past ten years two techniques have revolutionized the study of Ca2+ involvement in cell physiology: measurement of Ca2+ concentrations using ratiometric, Ca2+ specific fluorescent indicators such as fura-2; and the rapid increase in concentration of cellular substrates such as ATP, cGMP, etc. from a biologically inert or 'caged' form by flash-photolysis techniques. The objective of this proposal is to use rapid photochemical control of divalent cation levels to characterize the mechanism and regulation of cellular physiological processes. Ca2+-specific photolabile chelators will be developed and used to manipulate intracellular Ca2+ concentrations independently of other effectors, such as Mg2, ATP, GTP, etc. so that the regulatory roles that these species have on Ca2+-dependent cell physiology can be defined. Additionally, a Mg2+-specific photolabile chelator will be synthesized. Since Mg2+ is a necessary co-factor in nucleotide-dependent processes, the rapid photochemical release of the cation will be used to initiate these processes and characterize the role of Mg2+/nucleotide complexes in, for example, the mechanisms of muscle contraction and secretion. These studies will extend those already performed with DM-nitrophen, a photolabile chelator based on EDTA, developed by the applicant, which binds both Ca2+ and Mg2+ with high affinity. The basis of this proposal is a new Ca2+-specific photolabile chelator called nitrophenyl-EGTA, which has been recently synthesized. This caged Ca2+ will be used, in combination with fluorescent Ca2+ indicators, to study the kinetics and regulation of secretion events in melanotrophs and of contraction in cardiac, and smooth muscle because Ca2+ is the key intracellular second messenger in these systems. Many of these processes are distributed in pathological states. Before an adequate description of these disease states can be given, a more complete understanding of non-disease states should be accomplished. The proposed studies will contribute to a greater understanding of the normal functioning of these processes.